Necroptosis molecular mechanisms: Recent findings regarding novel necroptosis regulators

Abstract

Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein-protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis.

Fig. 1. Molecular mechanism of necroptosis.

TNF ligation induces complex I formation, which is composed of TRADD, TRAF2, cIAP1/2, RIPK1, TAK1, LUBAC, and the IKK complex, resulting in the activation of the NF-κB signaling pathway. When NF-κB target protein synthesis is inhibited by cycloheximide treatment, complex II a, consisting of TRADD, FADD, and caspase-8, is activated. Caspase-8, activated as part of complex II a, induces apoptosis through the cleavage of downstream molecules. The inhibition of RIPK1 ubiquitination or cytotoxicity-induced inhibition of phosphorylation, the early steps of TNF signaling, results in the induction of complex II b, which is composed of RIPK1, FADD, and caspase-8. Complex II b activation results in the induction of apoptosis through activated caspase-8. When caspase-8 is inhibited, RIPK1 and RIPK3 form necrosome complexes through homotypic interactions with RHIM, resulting in the activation of MLKL through a phosphorylation cascade. Phosphorylated MLKL undergoes oligomerization and migrates to the plasma membrane where it induces necroptosis by initiating membrane rupture or regulating ion flux. Death ligands, including FasL and TRAIL, initiate necroptosis by inducing necrosome complex formation. LPS, poly(I:C), double-stranded RNA, and viral RNA activate necroptosis by TRIF-mediated necrosome complex formation. Viral RNA or cellular endogenous RNA binding to ZBP1 results in RIPK1-independent necroptosis through the ZBP1–RIPK3 complex. Necroptosis factors are strictly regulated by ubiquitination, phosphorylation, glycosylation, and protein–protein interactions.

Fig. 2. Schematic overview of human RIPK1, RIPK3, and MLKL posttranslational modifications.

Overview of RIPK1, RIPK3, and MLKL posttranslational modifications, indicating the amino acid sites for ubiquitination, phosphorylation, and glycosylation with the enzymes have been reported to date.